Small-molecule Hsp90 Inhibitors

ABSTRACT

Purine scaffold Hsp90 inhibitors are useful in therapeutic applications and as radioimaging ligands.

STATEMENT OF RELATED CASES

This application is a continuation-in-part of U.S. application Ser. No.12/939,807, filed Nov. 4, 2010, which is a continuation of U.S.application Ser. No. 11/814,506 filed on Jul. 23, 2007, now U.S. Pat.No. 7,834,181, which is a national phase of PCT/US2006/003676, filedFeb. 1, 2006, which claims the benefit of U.S. Provisional PatentApplication No. 60/649,322 filed Feb. 1, 2005, all of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

This application relates to compounds effective as inhibitors of Hsp90,and to the use of such molecules in therapeutic applications. Themolecules of the invention are useful in therapeutic applications and asradioimaging ligands.

BACKGROUND OF THE INVENTION

The chaperone heat shock protein 90 (Hsp90) is an emerging target incancer treatment due to its important roles in maintainingtransformation and in increasing the survival and growth potential ofcancer cells. Hsp90 function is regulated by a pocket in the N-terminalregion of the protein that binds and hydrolyzes ATP. Occupancy of thispocket by high affinity ligands prevents the dissociation of Hsp90client proteins from the chaperone complex and as a consequence, thetrapped proteins do not achieve their mature functional conformation andare degraded by the proteasome. Protein clients of Hsp90 are mostlykinases, steroid receptors and transcriptional factors involved indriving multistep-malignancy and in addition, mutated oncogenic proteinsrequired for the transformed phenotype. Examples include Her2, Raf-1,Akt, Cdk4, cMet, mutant p53, ER, AR, mutant BRaf, Bcr-Abl, Flt-3, Polo-Ikinase, HQF-I alpha and hTERT. Degradation of these proteins by Hsp90inhibitors leads to cell-specific growth arrest and apoptosis in cancercells in culture, and to tumor growth inhibition or regression in animalmodels. One such inhibitor, 17-allyl-amino-desmethoxy-geldanamycin (17AAG, FIG. 1A) has entered clinical trials in cancer patients in the USand UK and has shown early evidence of therapeutic activity whenadministered alone or in combination with docetaxel. Despite these earlypromising results, 17AAG has several potential limitations. Mostprominent are its limited solubility and cumbersome formulation. It alsoexhibits dose and schedule dependent liver toxicity believed to becaused by the benzoquinone functionality. Radicicol (RD, FIG. 1B) astructurally unrelated natural product, has biological activity similarto that of 17AAG but is not hepatotoxic, yet no derivative of this classhas made it into clinic.

Making use of the peculiar bent shape of Hsp90 inhibitors and ofexistent Hsp90 crystal data, purine-scaffold derivatives with Hsp90inhibitory activities have been designed. PCT Application No.WO02/36075, which is incorporated herein by reference identified ageneralized structure for purine scaffold inhibitor class of compoundswith the formula:

The first synthesized derivative of this class, PU3 (FIG. 1C), boundHsp90 with moderate affinity and elicited cellular effects that mimic17AAG addition. Preliminary efforts focused at improving the potency ofthis agent have mostly focused on modifying the left side of thescaffold and have led to the synthesis of several compounds withimproved activity in both biochemical and cellular assays. One suchcompound, PU24FC1 (FIG. 1D) is a potent and selective inhibitor of tumorHsp90 and exhibits anti-tumor activities in both in vitro and in vivomodels of cancer. Other purine-scaffold compounds with higher potencyover PU24FC1 in in vitro models of cancer have subsequently beendisclosed. (See, PCT Patent Publication No. WO03/037860, which isincorporated herein by reference.) Although a significant number ofderivatives has been created by these combined efforts, the nature andposition of substituents on the right side aryl moiety (X₂ in thestructure above) and nature of the linker between the two rings have notbeen sufficiently investigated. The present invention provides a classof Hsp90 inhibitors with enhanced activity as compared to previouslyknown compounds and a class of inhibitors with differential selectivityand activity for Rb normal versus Rb defective cells.

SUMMARY OF THE INVENTION

In accordance with the present invention, Hsp90 inhibitors are providedhaving the formula:

left side-linker−right side

wherein(a) the left side has the formula of the left side in the PU family ofpurine scaffold inhibitor class of compounds:

in which:

X₆ is NH₂;

X₃ is hydrogen or halogen, for example F or Cl or Br; and

X₁ is hydrogen,

-   -   a C₁ to C₁₀ alkyl, alkenyl, or alkynyl group,    -   a saturated or unsaturated moiety containing up to 10 carbon        atoms and at least one heteroatom, or    -   a targeting or labeling moiety connected to the left side at N9,        (b) the linker is selected from the group consisting of CH₂, O,        NH and S, and        (c) the right side has the formula:

-six-membered aryl ring-(X₂)(X₄)(X₅)

wherein

X₂ is disposed at the two-position of the aryl ring and is halogen,alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl, pyrollyl, optionallysubstituted aryloxy, alkylamino, dialkylamino, carbamyl, amido,alkylamido dialkylamido, acylamino, alkylsulfonylamido, trihalomethoxy,trihalocarbon, thioalkyl, SO₂-alkyl, COO-alkyl, NH₂, OH, CN, SO₂X₅, NO₂,NO, C═S—R₂, NSO₂X₅, C═OR₂, where X₅ is F, NH₂, alkyl or H, and R₂ isalkyl, NH₂, NH-alkyl or O-alkyl; and

X₄ and X₅, which may be the same or different, are disposed at the 4 and5 positions of the aryl ring,

-   -   wherein X₄ and X₅ are selected from halogen, alkyl, alkoxy,        halogenated alkoxy, hydroxyalkyl, pyrollyl, optionally        substituted aryloxy, alkylamino, dialkylamino, carbamyl, amido,        alkylamido dialkylamido, acylamino, alkylsulfonylamido,        trihalomethoxy, trihalocarbon, thioalkyl, SO₂.alkyl, COO-alkyl,        NH₂ OH, CN, SO₂X₅, NO₂, NO, C═SR₂ NSO₂X_(5,), C═OR₂, where X₅ is        F, NH2, alkyl or H, and R₂ is alkyl, NH₂, NH-alkyl or O-alkyl,        C₁ to C₆ alkyl or alkoxy; or    -   wherein X₄ and X₅ taken together have the formula        —O—(CH₂)_(n)—O—, wherein n is an integer from 0 to 2.

The Hsp90 inhibitors can be used for therapeutic application in thetreatment of cancer and other conditions where the cells depend on hsp90activity for cell growth or maintenance. Radiolabeled Hsp90 inhibitorsof the invention are useful as radiotracers for imaging tumors thatexpress Hsp90.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and B shows the structure of prior art Hsp90 inhibitors 17-AAGand radicicol.

FIGS. 1C and 1D shows the structure of prior art purine-scaffold Hsp90inhibitor PU3 and PU24FC1.

FIG. 2 shows a general structure of embodiments of a purine-scaffoldHsp90 inhibitor in accordance with the invention in which the left sideis an adenine (N at 1 and 3 positions).

FIG. 3 A-Q shows the structures of various purine-scaffolded Hsp90inhibitors in accordance with the present invention.

FIG. 4 shows the structure of an exemplary Hsp90 inhibitor connected toa targeting moiety via a connector.

FIGS. 5A-D shows synthetic schemes for making compositions in accordancewith the invention.

FIGS. 6A and B compare the effectiveness of PU24FC1and a compound of theinvention against various small cell lung cancer cells lines.

FIGS. 7A-D show experimental results for compounds in accordance withthe invention, as compared to PU24FC1.

FIG. 8 shows inhibition of cell growth and induction of cell death inSCLC cells highly resistant to conventional chemotherapy using Example 9in accordance with the invention.

FIGS. 9A-D show a comparison of Example 9 in accordance with theinvention and prior art compounds for efficacy against small cell lungtumors.

FIGS. 10A and B show pharmacodynamic effects of Compound 6 whenadministered to MDA-MB bearing mice.

FIGS. 11 A and B show enhancement of radiation sensitivity in gliomacells after pretreatment with to Compounds 9 or 8, respectively.

FIG. 12 shows example displacement curves for [¹³¹I]-Compound 9 and[¹³¹I]-Compound 9.

FIG. 13 shows saturation curves [¹³¹I]-Compound 8 and [¹³¹I]-Compound 9.

FIG. 14 shows the ratio of [¹³¹I]-Compound 8 and [¹³¹I]-Compound 9 intumor and muscle and tumor and blood 4 and 24 hours afteradministration.

DETAILED DESCRIPTION OF THE INVENTION Compositions of the Invention

The present application provides small molecule Hsp90 inhibitors thatare purine-scaffold derivatives of adenine with the general structure:

left side-linker−right side

wherein(a) the left side has the formula of the left side in the PU family ofpurine scaffold inhibitor class of compounds:

in which:

X₆ is NH₂;

X₃ is hydrogen or halogen, for example F or Cl or Br; and

X₁ is hydrogen,

-   -   a C₁ to C₁₀ alkyl, alkenyl, or alkynyl group,    -   a saturated or unsaturated moiety containing up to 10 carbon        atoms and at least one heteroatom, or    -   a targeting or labeling moiety connected to the left side at N9,        (b) the linker is selected from the group consisting of CH₂, O,        NH and S, and        (c) the right side has the formula:

-six-membered aryl ring-(X₂)(X₄)(X₅)

wherein

X₂ is disposed at the two-position of the aryl ring and is halogen,alkyl, alkoxy, halogenated alkoxy, hydroxyalkyl, pyrollyl, optionallysubstituted aryloxy, alkylamino, dialkylamino, carbamyl, amido,alkylamido dialkylamido, acylamino, alkylsulfonylamido, trihalomethoxy,trihalocarbon, thioalkyl, SO₂-alkyl, COO-alkyl, NH₂, OH, CN, SO₂X₅, NO₂,NO, C═S R_(2,) NSO₂X_(5,), C═OR₂, where X₅ is F, NH₂, alkyl or H, and R₂is alkyl, NH₂, NH-alkyl or O-alkyl; and

X₄ and X₅, which may be the same or different, are disposed at the 4 and5 positions of the aryl ring,

-   -   wherein X₄ and X₅ are selected from halogen, alkyl, alkoxy,        halogenated alkoxy, hydroxyalkyl, pyrollyl, optionally        substituted aryloxy, alkylamino, dialkylamino, carbamyl, amido,        alkylamido dialkylamido, acylamino, alkylsulfonylamido,        trihalomethoxy, trihalocarbon, thioalkyl, SO₂.alkyl, COO-alkyl,        NH₂ OH, CN, SO₂X₅, NO₂, NO, C═SR₂ NSO₂X_(5,), C═OR₂, where X₅ is        F, NH2, alkyl or H, and R₂ is alkyl, NH₂, NH-alkyl or O-alkyl,        C₁ to C₆ alkyl or alkoxy; or    -   wherein X₄ and X₅ taken together have the formula        —O—(CH₂)_(n)—O—, wherein n is an integer from 0 to 2.

As would be understood by persons skilled in the art, atoms named aspart of the compound have an appropriate number of bonds to adjacentatoms to satisfy valency requirements for the particular atom.

In some embodiments of the invention, the left side contains twonitrogen atoms at the 1 and 3 positions.

The six-membered aryl group of the right side may be phenyl or mayinclude one or more heteroatoms. For example, the six-membered arylgroup may be a nitrogen-containing aromatic heterocycle such aspyrimidine.

In specific preferred embodiments of the composition of the invention,the groups X₄ and X₅ taken together have the formula —O—(CH₂)_(n)—O—,wherein n is an integer from 0 to 2, preferably 1 or 2. One of theoxygens is bonded at the 5′-position of the aryl ring and the other atthe 4′ position. In other specific embodiments of the invention, thesubstituents X₁ comprise alkoxy substituents, for example methoxy orethoxy, at the 4′ and 5′-positions of the aryl ring.

In specific embodiments of the invention, the substituent X₂ is ahalogen.

In specific embodiments, X₂ is NH₂, alkylamino or dialkylamino.

In specific embodiments, X₁ is an amino alkyl moiety, optionallysubstituted on the amino nitrogen with one or two carbon-containingsubstituents selected independently from the group consisting of alkyl,alkenyl and alkynyl substituents, wherein the total number of carbons inthe amino alkyl moiety is from 1 to 10.

In specific embodiments of the invention, the linker is S. In otherspecific embodiments of the invention, the linker is CH₂.

In specific embodiments of the invention, X₁ is a pent-4-ynylsubstituent. In other specific embodiments of the invention, X₁ containsa heteroatom, for example nitrogen. A preferred X₁ group that increasesthe solubility of the compound relative to an otherwise identicalcompound in which X₁ is H or pent-4-ynyl is —(CH₂)_(n)—N—R₁₀R₁₁ or—(CH₂)_(n)—N⁺—R₁₀R₁₁R₁₂, where m is 2 or 3 and where R_(10.12) areindependently selected from hydrogen, methyl, ethyl, ethene, ethyne,propyl, isopropyl, isobutyl, ethoxy, cyclopentyl, an alkyl group forminga 3 or 6-membered ring including the N, or a secondary or tertiary amineforming a 6-membered ring with the nitrogen. In specific examples, R₁₀and R₁₁ are both methyl, or one of R₁₀ and R_(n) is methyl and the otheris ethyne. When X₁ contains a quaternary nitrogen, a pharmaceuticallyacceptable counterion is also present.

FIG. 3A shows exemplary structures in accordance with the presentinvention. The structures are based on one of two right side arylsubstitution patterns in which the 4′ and 5-substituents are either abridging substituent or dihydroxy/alkoxy. The left side adenine hasvarying substituents (Cl, F or H) at the 2-position (X₃), and choicesfor the N9 substituent X₁ are listed. FIGS. 3 B-Q shows additionaloptions for specific structures in accordance with the invention.

In some embodiments of the invention, X₁ includes a cyclic portion. Insome embodiments, this cyclic portion includes one or more heteroatoms.The cyclic portion may be a three membered ring. The cycle portion maybe a six membered ring.

Synthesis of Compositions in Accordance with the Invention

Synthesis of sulfur linker derivatives can be achieved using theprocedures as outlined in Scheme 1 (FIG. 5A). Formation of a sulfur linkbetween adenine and the phenyl ring (linker=S) may be obtained either bynucleophilic attack of the arylthiolate anion on 8-bromoadenine (Step a,Scheme 1) or by the copper catalyzed coupling of aryliodides withmercaptoadenine (Step b, Scheme 1). Our developed method for theformation of 8-arylsulfanyl adenine derivatives (5) from8-mercaptoadenine (3) and aryl iodides (4) uses Cul/neocuproine ascatalyst and NaOt-Bu/DMF as the base/solvent combination. The reactionoccurs in anhydrous DMF at 110° C. under nitrogen to generate theproducts in good yields. If bromine or chlorine is present on the arylmoiety, the coupling requires milder basic condition and utilizes Na₃PO₄or K₂CO₃ instead of NaOt-Bu. Although less attractive due tothiophenols' limited commercial availability, stench and tendency toquickly oxidize, coupling of 8-bromoadenine (1) with thiophenols in thepresence of a base will also be used. When not commercially available,thiophenols can be generated by a modified Leuckart thiophenol reactionstarting from the corresponding aryl amine. In a first step, aryl amineswill be converted via the aryl diazonium salt to aryl xanthates whichafford thiophenols on reduction with LiAlH₄ or on warming with a basesolution. The 8-arylsulfanyl adenines 5 obtained in these couplingreactions will be further alkylated at the position 9-N with pent-4-ynylor the corresponding -bromoalkylalcohols. Their introduction will becarried out using a Mitsunobu type reaction between the alcohol and therespective 8-arylsulfanyl adenines in toluene/CH₂Cl₂ to result in thecorresponding 9-N-alkyl-8-arylsulfanyl adenines. Formation of the 3-Nand 7-N isomers is likely to be observed (˜0 to 30%), however, thesebyproducts will be removed by column chromatography (differences in Rfare considerable). Treatment of 5 with base and an alkylating agenttends to result in higher percentage of 3- and 7-N isomers compared toalcohol treatment under the Mitsunobu conditions. Heating these bromineswith amines in DMF will generate the desired products. Phosphate saltswill be further made for in vivo administration of selected compounds toimprove their water solubility.

Synthesis of methylene linker derivatives can be accomplished using theprecoures outlined in Scheme 2 (FIG. 5B). If the linker is methylene(linker=CH₂), synthesis will commence with coupling of the commerciallyavailable 2,4,5,6-tetraaminopyrimidine sulfate (9) with the acidfluoride (8) of the corresponding carboxylic acids. We have previouslydetermined this to be the only coupling method that in our hands givesthe product in high yields. Acid fluorides are generated by treating thecorresponding carboxylic acids with cyanuric fluoride and pyridine inCH₂Cl₂. Following a quick water wash, the resulted acid fluorides areused in the next step without further purification. To make thenecessary 2-chloro, bromo or iodo-4,5-derivatized phenyl acetic acids wehave determined optimal reaction conditions. We have identified thattreatment of 4,5-derivatized phenyl acetic acids with ICl/AcOH; NBS; orHCl/t-BuOOH gives solely ortho-iodinated; brominated; or chlorinatedproduct, respectively in high yields. The amides resulted from thepyrimidine-acid fluoride couplings will be cyclized by heating inalcoholic NaOMe. Transformation of the C2-amino group to fluorine(X₃═NH₂ to F) will be conducted by diazotization-fluorodediazoniation ofthe amino derivative in HF/pyridine in the presence of NaNO₂ to yieldthe corresponding adenine derivatives 10. This reaction givessignificantly higher yields over the method using HBF₄/iso-amyl nitrite,likely to be due at least in part to the more anhydrous nature of thissolvent system which reduces the proportion of hydrolysis. Furtheralleviation will be conducted using the reaction sequence mentioned inScheme 1 to result in derivatives 12.

Several derivatives were prepared by the introduction of fluorine atposition C2 of the adenine moiety (Scheme 3, FIG. 5C). We and othershave previously determined that fluorine in this position in generalincreased the solubility and/or potency of the resulting purines. Forthe preparation of such derivatives, synthesis commenced with thecondensation of the commercially available 2,4,5,6-tetraaminopyrimidinesulfate (14) with carbon disulfide in a refluxing solution of NaHCO₃ inaqueous Et0H.¹⁷ The resulting 2-amino-8-mercaptoadenine (15) was furthercoupled with aryl iodides in the presence of NaOt-Bu and CuI in ethyleneglycol to give 2-amino-8-arylsulfanyl adenines 16. The reaction did notproceed using the method published by He et al as 15 did not dissolve inDMF or most organic solvents for that matter. Unsaturated X₁ chains atposition 9-N were introduced at this stage to result in the2-amino-9-N-alkyl-8-arylsulfanyl adenines 17. The reaction required theuse of K₂CO₃ or Cs₂CO₃ and an alkylating agent in DMF due to the poorsolubility of 16 in the Mitsunobu reaction solvent. If further,introduction of chlorine at X₂ on the aryl moiety was desired, compounds17 were subjected to HCl/t-BuOOH to result preferentially inorto-chlorinated compounds (18). Transformation of the C2-amino group tofluorine (X₄═NH₂ to F) was conducted bydiazotization-fluorodediazon-iation of the amino derivative inHF/pyridine in the presence of NaNO₂ to yield the corresponding2-fluoro-9-N-alkyl-8-arylsulfanyl adenine derivatives 21. This reactiongave significantly higher yields over the previously published methodusing HBF₄/iso-amyl nitrite, likely to be due at least in part to themore anhydrous nature of this solvent system which reduces theproportion of hydrolysis. Synthesis of derivatives containing both anunsaturated chain at 9-N (i.e. pent-4-ynyl) and fluorine at the C2position of the adenine moiety (X₃═F) required a different strategy toavoid fluorine addition to the triple bond. Compound 16 was firstsubjected to introduction of fluorine at C2 to result in thecorresponding 2-fluoro-8-arylsulfanyl adenine 19. Surprisingly,conducting the reaction at room temperature resulted in addition offluorine at both C2 and C6 of the purine moiety. This problem wasaverted by lowering the reaction temperature to −40° C. Addition offluorine at position C2 significantly increased the solubility of thesepurines in organic solvents. Thus, further alkylation could be easilyconducted using the Mitsunobu reaction in toluene: CH₂Cl₂ to result inderivatives 20. Introduction of chlorine (X₂═Cl), again with HCl/t-BuOOHled to formation of 21. Alternatively, synthesis of2-fluoro-9-N-alkyl-8-arylsulfanyl adenines 20 was started with thecommercially available 2-fluoroadenine (22) (Scheme 3). This reagent wasfirst 9-N alkylated by Cs₂CO₃/tosylate treatment followed by8-bromination with N-bromosuccinimide (NBS) 19 to result in2-fluoro-9-N-alkyl-8-bromo adenine 23. Its coupling with thiophenolsgenerated the 2-fluoro-9-N-alkyl-8-arylsulfanyl adenines 20.

Syntheses of 8-arylsulfoxyl adenine derivatives 27 and 8-arylsulfonyladenine derivatives 28 (Scheme 4, FIG. 5D) were previously reported byLlauger et al. Briefly, synthesis started with the preparation of Q-NH₂triphenylphosphine-protected 9-N-alkyl-8-arylsulfanyl adeninederivatives 24 in a two-step (alkylation-protection) one-pot reactionfrom the corresponding 8-arylsulfanyl adenines 10 using the Mitsunobuconditions 13b followed by addition of an excess of PPh3 anddi-tert-butyl azodicarboxylate (DBAD). Oxidation of 24 with OXONE® inthe presence of alumina 22 allowed for monitoring the reaction to eithersulfoxide (25) or sulfone (26). Deprotection of the C6-NH₂triphenylphosphine group was conveniently conducted in refluxingAcOH/EtOH to result in good yields in the corresponding 8-arylsulfoxyladenine derivatives 27 and 8-arylsulfonyl adenine derivatives 28. Thelater could be chlorinated at C2 (X₂=Cl) by HCl/t-BuOOH treatment toresult in derivatives 29.

Using these methods, the compositions described in this application wereprepared. These compositions include various compositions in accordancewith the invention, as well as comparative examples.

Biological Testing

Compounds synthesized above were tested in a biochemical assay, and alsoin cellular assays that probe for cellular fingerprints of Hsp90inhibition. The biochemical assay tests competitive binding of compoundsto recombinant Hsp90a protein and also Hsp90 found in cell specificcomplexes, and uses a fluorescence polarization method. When using celllysates instead of recombinant protein, the assay measures binding toaverage. Hsp90 population found in cell specific complexes. The cellularassays measure two specific biological effects observed upon addition ofknown Hsp90 inhibitors to cancer cells: (a) degradation of the tyrosinekinase Her224 and (b) mitotic block in Rb-defective cells.

Overexpression of the receptor tyrosine kinase Her2 in SKBr3 breastcancer cells leads to Akt activation which in turn promotes cellsurvival. Hsp90 uniquely stabilizes Her2 via interaction with its kinasedomain and an Hsp90 inhibitor induces Her2 degradation by disrupting theHer2/Hsp90 association. We have previously reported a fast microliterimmunoassay able of quantifying cellular levels of Her2 following drugtreatments. This assay is used here to differentiate theHer2-degradative potential of the above synthesized purines. Hsp90inhibitors are also known to cause cells lacking functional RB toprogress normally through Gl and arrest in mitosis. Thus, another assayused here to test cellular Hsp90 inhibition relies on assessing theanti-mitotic potential of synthesized purines. The assay is aniicrotiter immunoassay and uses an antibody against a mitoticallyphosphorylated form of nucleolin to detect cells in mitosis. Thisantibody (Tg-3), originally described as a marker of Alzheimer'sdisease, is highly specific for mitotic cells, Tg-3 immunofluorescencebeing >50-fold more intense in mitotic cells than in interphase cells.In addition, the cytotoxicity of these agents against SKBr3 breastcancer cells was determined. A selected number of most active purineswere also tested for possible toxicity against a normal cell line, renalproximal tubular epithelial cells (RPTEC).

Table 1 shows compounds that were tested for biological activity. Thesecompounds are identified by an example number in the table, and arereferred to herein by that number as

Example or Compound______. In each case, for the compounds tested, thevariable ring members in the left side were both nitrogen.

Table 2 shows results for EC₅₀ Hsp90α, IC₅₀ for Her2 degradation, andIC₅₀ for growth inhibition in SKBr3 breast cancer cells. All values inTable 2 in μM and represent an average of 3 measurements. As can beseen, the compositions tested all show substantial activity, and in manycases activity at nanomolar concentrations.

TABLE 1 Example X₄, X₅ X₂ linker X₃ X1 1 (PUH58) —OCH₂O— Br S Hpent-4-ynyl 2 Cl, Cl Cl S H pent-4-ynyl 3 4-Cl, Cl S H2-isopropoxy-ethyl 5-methoxy 4 Cl, Cl Cl S H H 5 —OCH₂O— Br S HN-isopropyl-n- propylamine 6 (PUDZ2) —OCH₂O— Br CH₂ F pent-4-ynyl 7(PUDZ3) —OCH₂O— Cl CH₂ F pent-4-ynyl 8 (PUDZ8) —OCH₂O— I CH₂ FN-isopropyl-n- propylamine 9 (PUH71) —OCH₂O— I S H N-isopropyl-n-propylamine 10 —OCH₂O— I S H pent-4-ynyl 11 —OCH₂O— I CH₂ F pent-4-ynyl12 4,5-dimethoxy I CH₂ F pent-4-ynyl 13 4,5-dimethoxy Br CH₂ Fpent-4-ynyl 14 4,5-dimethoxy Cl CH₂ F pent-4-ynyl 15 —OCH₂O— I CH₂ HN-isopropyl-n- propylamine Comp 1 3′,4′,5′- Cl CH₂ F pent-4-ynyl(PU24FCl) trimethoxy Comp 2 3′,4′,5′- Cl S F pent-4-ynyl trimethoxy

TABLE 2 EC₅₀ Hsp90-α IC₅₀ Her2 IC₅₀ SKBr3 Anti- Example (μM) (μM) (μM)mitotic 1  0.03 ± 0.005  0.3 ± 0.05  0.2 ± 0.01 Yes .0508 ± .004  0.365± 0.0 45 0.300 ± 0.05 Yes 2  8.5 ± 0.1  58 ± 1.4 48.4 ± 2.5 No 3  5.0 ±1.1 58.3 ± 2.2  16.1 ± 0.7 No 4 15.4 ± 2.2 47.8 ± 3.2  36.0 ± 0.2 Yes 50.0388 ± 0.003 0.205 ± 0.015  0.142 ± 0.022 Yes 6 0.0565 ± 0.002 0.210 ±0.01   0.215 ± 0.055 Yes 7 0.0772 ± 0.001 0.300 ± 0.015  0.250 ± 0.030Yes 8 0.0504 ± 0.004 0.080 ± 0.010 Yes 9 0.010.8 ± 0.002  0.100 ± 0.010 0.090 ± 0.002 Yes 11 0.0223 ± 0.002 0.090 ± 0.01  0.090 ± 0.03 Yes12 >15 >50 >50 No 13 >15 >50 55.0 ± 2.3 No 14  4.6 ± 0.02 30.0 ± 1.0 20.6 ± 6.7 No Comp 2 0.12 ± 0.3 1.3 ± 0.4  1.8 ± 0.2 Yes

Several active derivatives were tested for specificity towardstransformed cells (Table 3). Binding affinities of selected compoundsfor average population Hsp90 complexes found in normal tissues (brain,lung and heart) and in addition, their cytotoxicities against RPTECnormal cells were determined. Compounds were found to bind Hsp90 fromnormal tissues with 2- to 3-log weaker affinities when compared to Hsp90from SKBr3 cells. This specificity translated into 5 to 100-foldselectivity (column 10, Table 3) in inhibiting the growth of transformedcells compared to cultured normal epithelial cells (RPTEC tested). Nocell death was observed in the purine-scaffold treated RPTEC cells evenat the highest tested concentrations. Selectivity was also observedbetween SKBr3 cells and MRCS normal lung fibroblasts for compounds 9 and10.

TABLE 3 EC₅₀ Ec₅₀ EC₅₀ EC₅₀ Hsp90 Hsp90 Hsp90 Hsp90 Brain/ Lung/ Heart/IC₅₀ RPTEC/ Ex. Brain lung heart SKBr3 SKBr3 SKBr3 SKBr3 RPTEC SKBr3 140.2 ± 25.3 14.7 ± 1.7  65.3 ± 9.9  0.02 ± 0.06 2000 735 3265  4.1 ± 0.920.5 6 ND  5.20 ± 0.210 13.30 ± 0.210 .0388 ± 0.003 ND 134 343 ND ND 9ND 2.40 ± 0.24 6.90 ± 0.15 0.0504 ± 0.004  ND 48 140 ND ND 10 ND 2.20 ±0.40 6.00 ± 0.20 0.0161 ± 0.001  ND 136 370 ND ND Comp2 26.7 ± 7.7  217± 50  53.1 ± 20.5 0.09 ± 0.03  298 2400 590 28.1 ± 2.2 15.6

A compound in accordance with the invention having the structure shownin FIG. 3B (Compound 1) and PU24FC1, (Comparative 1, FIG. 1D) weretested for activity against 7 different small cell lung cancer celllines (NCI-H69, NCI-H146, NCI-H209, NCI-H187, NCI-N417, NCI-H510 andNCI-H256) obtained from the American Type Culture Collection (Manassas,Va.). Both compounds exhibited binding to cellular Hsp90 as determinedby homogenous fluorescence polarization. However, the concentrationrequired of the compound of the invention was about 1 order of magnitudeless than for PU24FC 1. (Table 4) Antiproliferative effects of the twocompounds were observed in the SCLC cell lines, and the percentage ofapoptotic cells were determined. As shown in FIG. 6A (PU24FC1) and B(Example 1), both compounds induced apoptosis in each of the SCLC cellslines, but the composition of the invention does so at a lowerconcentration. Both PU24FC1 and its higher potency derivative, Example1, inhibit the growth and cause significant cytotoxicity against allSCLC cell lines. At concentrations of Hsp90 inhibitors in excess of 4-12mM and 0.25-1 mM, respectively, 50-100% of the starting cell populationsare dead at 72 h or 96 h post-treatment. As shown in Table 4 and FIGS. 6A and B, the 10 to 20-fold difference in binding for the two drugscorrelate well with the their anti-proliferative potencies, suggestingthat cell death in these cells is a direct result of Hsp90 inhibition.

TABLE 4 IC₅₀ IC₉₀ EC₅₀ IC₅₀ IC₉₀ EC₅₀ IC₅₀ EC₅₀ Comp 1 Comp 1 Comp 1Examp 1 Examp 1 Exampl 1 Comp 1/Ex 1 Comp 1/Ex 1 NCI-H526 4.2 ± 0.6  7.4± 0.1 0.229 ± 0.04 0.44 ± 0.04 0.83 ± 0.05 0.023 ± 0.02 9.5  9.6NCI-N417 3.5 ± 0.3  5.3 ± 0.4 0.592 ± 0.13 0.33 ± 0.03 0.61 ± 0.01 0.038± 0.01 10.6 15.4 NCI-H146 5.8 ± 0.2 10.4 ± 0.2 0.574 ± 0.22 0.27 ± 0.040.49 ± 0.02 0.049 ± 0.02 21.5 13.1 NCI-H187 6.5 ± 0.3 12.5 ± 0.5 ND 0.64± 0.02  1.2 ± 0.03 ND 8.3 ND NCI-H209 9.5 ± 0.5 11.6 ± 0.4 0.347 ± 0.060.50 ± 0.05 0.65 ± 0.04 0.021 ± 0.04 19.0 16.0 NCI-H510 10.7 ± 0.1  13.4± 0.6 ND 0.76 ± 0.02 0.90 ± 0.09 ND 14.1 ND NCI-H69 2.5 ± 0.2  4.0 ± 0.10.350 ± 0.1  0.17 ± 0.02 0.25 ± 0.03 0.019 ± 0.01 14.7 17.8

FIGS. 3B-E show the structure of four compounds (examples 1, 5, 6 and 7,respectively) in accordance with the invention made using the methods ofSchemes 1 and 2. Where the 9N-alkyl chain is pent-4-ynyl, 5 and 10 weredirectly alkylated with using the Mitsonobu reaction and thecorresponding alcohol. FIG. 7A-D shows test results for these compoundswhen compared to the prior art compound of FIG. 1D (Comp 1). FIG. 7Ashows results when exponentially growing SKBr3 cancer cells were seededinto 96-well plates and incubated in medium containing either of vehiclecontrol (DMSO) or the test compound (four replicate wells per assaycondition) at the above indicated concentrations for 72 h at 37° C.

The antiproliferative effects of test compounds were evaluated using theCellTiter-Glo® Luminescent Cell Viability Assay kit from PromegaCorporation. FIG. 7B shows results when exponentially growing SKBr3cancer cells were seeded into 96-well plates and incubated in mediumcontaining either of vehicle control (DMSO) or the test compounds (fourreplicate wells per assay condition) at the above indicatedconcentrations for 24 h at 37° C. The Her2 degradation potential of PUswas evaluated using our Her2 blot procedure described in PCT PatentApplication Serial No. PCT/USO4/21297, which is incorporated herein byreference. FIG. 7C shows results when exponentially growing MDA-MB-468cancer cells were seeded into 96-well plates and incubated in mediumcontaining either of vehicle control (DMSO) or the test compounds (fourreplicate wells per assay condition) at the above indicatedconcentrations for 24 h at 37° C. The anti-mitotic potential ofcompounds was evaluated using our Tg3 blot. The resultedchemiluminescent signal was read with an ANALYST® AD microplate reader.FIG. 7D shows the apoptotic inducing potential of PUs, which wasassessed using caspase-3,7 activation as read-out. MDA-MB-468 cells(plated at 8,000/well) were plated in 96-well plates and treated for 24h with varying concentration of compounds. For the caspase-3,7 assay,following treatment cells are lysed and permeabilized with our in-housedeveloped buffer (10 mM HEPES pH. 7.5, 2 mM EDTA, 0.1% CHAPS, 0.1 mg/mlPMSF, COMPLETE™ Protease Inhibitor Mix, Roche) to make them accessibleto the caspase-3,7 substrate Z-DEVD-R110. This agent becomes highlyfluorescent upon cleavage by activated caspase-3,7 and subsequentrelease of rhodamine, thus the assay is a simple read-mix procedure. Theresulted fluorescence signal will be read using the SPECTRAMAX® GeminiXS (Molecular Devices) (ex. 485, em. 530). As is apparent from thesefigures, the four compositions of the invention all produced similarresults to one another, but were active at significantly lowerconcentrations than the comparison compound.

The compounds of FIGS. 3F and 3G (Examples 8 and 9) are water solublewhen in the acid addition salt form (solubility>5 mM in PBS) and havelow nanomolar potency against cancer cells. The pharmacokinetic andpharmacodymanic profiles, as well as the anti-cancer activity ofCompound 9 were tested in a model of small cell lung cancer. Small celllung carcinoma (SCLC) is a highly malignant tumor accounting for about20% of all lung cancers. It is a disease that metastasises early andwidely, accounting for the extremely poor prognosis of this tumor.Whereas it often initially responds well to chemotherapy, relapses occuralmost without exception, and they are usually resistant to cytotoxictreatment. We have shown that compound of FIG. 3G (Example 9) retainsits activity in H69AR, a SCLC cell line resistant to adriamycin as wellas in SKI-Chen, a cell line derived at MSKCC from a patient who failedto respond to every conventional therapy. The H69AR line was establishedfrom NCI-H69 cells that were grown in the presence of increasingconcentrations of adriamycin (doxorubicin) over a total of 14 months.The cell line is cross-resistant to anthracycline analogues includingdaunomycin, epirubicin, menogaril, and mitoxantrone as well as toacivicin, etoposide, gramicidin D, colchicine, and the Vinca alkaloids,vincristine and vinblastine, and expresses the multidrug resistanceprotein. Growth over 96 h was assessed. FIG. 8 shows a comparison ofresults for Compound 9 and PU24FC 1. Values below 0% represent celldeath of the starting population. As shown, the compound of theinvention is effective at a concentration about 20 times lower.

In vivo, Example 9 exhibits the tumor retention profile manifested byour early micromolar compound of FIG. 1D (Comp 1) as shown in FIGS. 9 Aand B. While Example 9 is rapidly cleared from plasma, with levelsundetectable after 6 h post-administration, it is retained in tumors atpharmacologic doses for more than 36 h. Such behavior translates inconsiderable downregulation for more than 36 h of Hsp90 client proteinsdriving transformation in this tumor. Proteins involved in growth andsurvival potential of the tumor, Raf-1 and Akt, are efficiently degradedor inactivated (pAkt) by one administered dose of Example 9. Inaddition, the agent induces significant apoptosis of the tumor asreflected by an increase in PARP cleavage. (FIG. 9C) This is the firstindication of apoptosis induced in vivo by an Hsp90 inhibitor.Concordantly, Example 9 efficiently inhibited the growth of this tumorin a manner comparable to 17AAG without toxicity to the host (FIG. 9D).

Mice xenografted with MDA-MB-468 human breast cancer tumors were treatedby intraperitoneal injection with Compound 5 at a dosage levels of 25,50, 75, 100 and 150 mg/kg. The concentration of Compound 5 in the tumor24 hours after administration was determined. As reflected in FIG. 10A,at all dosage levels a biologically active concentration of 300 nM wasobserved in the tumor, although it could not be detected in plasma.Compound 5 was further tested for its effects on the pharmacodynamicmarker Raf-1 kinase.

Hsp90 stabilizes this kinase and maintains it in ready-to-be-activatedconformation. Inhibition of Hsp90 leads to disruption of the complex andfurther ubiquitinylation and degradation of Raf-1 by the proteasome.Thus, Raf-1 degradation in tumors is a functional read-out of Hsp90inhibition. FIG. 10B shows % control levels of Raf-1 protein in mice towhich Compound 5 was administered. As shown, substantial decrease inRaf-1 levels was observed at all dosage levels. In contrast, no changein expression of PD kinase, a protein unaffected by Hsp90 inhibitors.

Observation of selective activity

Although the change of the linker structure between CH₂ and S did notresult in significant change in activity measured in the biologicalassays described above, this work has allowed the identification ofHsp90 inhibitors that demonstrate selective affinities for certainHsp90-client protein complexes. Compounds Example 1 and Comp 2 inducedHer2 degradation and inhibition of growth in SKBr3 cells, and alsoexhibited anti-mitotic activity in MDA-MB-468 cells, these eventsoccurring with similar potencies. However, among the moderate affinitybinders, derivatives were identified that degrade Her2 withcorresponding potencies but do not affect cell cycle distribution inRB-defective cells at similar concentrations. The Hsp90 client proteinof whose inactivation by Hsp90 inhibitors is responsible for the blockof these cells in mitosis is currently unknown. Due to their selectivityprofile, these derivatives may be useful pharmacological tools indissecting Hsp90-regulated processes.

A study comparing the activity of pairs of compounds differing only inthe nature of the linker indicated that in general, compounds with CH₂as the linker are antimitotic, while compounds with S as the linker arenot. Consistent with this observation, caspase 3,7 assays showed thatcompounds with a CH₂ linker induce apoptosis in Rb defective cells,while the S compound does not, This is indicative of a selectiveaffinity to hsp90 complexes in these cells. Both S and CH₂ compounds mayhave comparable affinity for hsp90 complexes that regulate cell growthand survival, regardless of Rb-type, while the S compounds are moreweakly bound to hsp90 complexes that regulate transition through mitosisin Rb-defective cells. As a result of this selectivity, S compounds aremore beneficial in the treatment of diseases/conditions where apoptotisis not desired. This would include neurodegenerative diseases, ischemia,inflammation, HIV and nerve regeneration.

Compositions Coupled to Targeting/Labeling Moieties

The compounds of the invention may be coupled via N9 to a targetingmoiety selected to specifically bind to a protein, receptor or markerfound on a target population of cells. The targeting moiety may be ahormone, hormone analog, protein receptor- or marker- specific antibodyor any other ligand that specifically binds to a target of interest, andis selected on the basis of the identity of the target. Particulartargeting moieties bind to steroid receptors, including estrogen andandrogen and progesterone receptors, and transmembrane tyrosine kinases,src-related tyrosine kinases, raf kinases and PI-3 kinases. Specifictyrosine kinases include HER-2 receptors and other members of theepidermal growth factor (EGF) receptor family, and insulin andinsulin-like growth factor receptors. Examples of specific targetingmoieties include estrogen, estradiol, progestin, testoterone, tamoxifenand wortmannin. Targeting moieties may also be antibodies which bindspecifically to receptors, for example antibodies which bind to Her2receptors as disclosed in International Patent Publications Nos.WO96/32480, WO96/40789 and WO97/04801, which are incorporated herein byreference.

FIG. 4 shows an exemplary structure of a composition of the inventionthat includes a targeting moiety connected to the nitrogen at the 9position of the purine via a connector. The linker may be any generallylinear chain which is of sufficient length permit the targeting moietyand the purine scaffold molecule to interact with their particulartargets when associated together, or may be a linker adapted forcontrolled cleavage once targeting has been accomplished. The linker maybe a C₄ to C₂₀ hydrocarbon chain, or may include intermediateheteroatoms such as 0 or N. Commonly, the linker is coupled to N-9 ofthe purine scaffold using the same synthetic mechanisms for addingsubstituents to the N-9 position. Terminal functional groups, such asamino or carboxyl groups, on the linker are used to form a bond withreactive sites on the selected targeting moiety.

In lieu of a targeting moiety, the compounds of the invention mayinclude a labeling moiety attached via a connector to the N9 position.Examples of labeling moieties include without limitation biotin. As inthe case of a targeting moiety, the connector is not critical instructure, and need only be of sufficient length so that the labelingmoiety does not interfere with the interaction of the purine scaffoldportion of the molecule with Hsp90.

Use of the Compositions of the Inventions

Because of their ability to bring about the degradation of proteinswhich are essential to cellular function, and hence to retard growthand/or promote cell death, the hsp90-binding compounds of the invention,with or without a targeting moiety, can be used in the therapeutictreatment of a variety of disease conditions. A suitable therapeutic isone which degrades a kinase or protein that is found in enhanced amountsor is mutated in disease-associated cells, or on which the viability ofsuch cells depends. The general role of HSP90 proteins in maintainingmalignancy in most cancer cells points to the importance of this targetin the development of anticancer agents. Thus, the therapeutic smallmolecules of the invention provide a novel modality for the treatment ofall cancers that require or are facilitated by an HSP90 protein. Forexample, the compositions of the invention can be used in the treatmentof a variety of forms of cancer, particularly those that overexpressHer2 or mutated or wild type steroid receptors, or that lack functionalRB protein. Such cancers may include but are not limited to breastcancer, small cell lung cancer, amyelocytic leukemia, vulvar cancer,non-small cell lung cancer, colon cancer, neuroblastoma and prostatecancer. In addition, the compositions of the invention can be used inthe treatment of other diseases by targeting proteins associated withpathogenesis for selective degradation. Examples of such targetableproteins include antigens associated with autoimmune diseases andpathogenic proteins associated with Alzheimer's disease.

The compositions of the invention exhibit the ability to degradespecific kinases and signaling proteins. Furthermore, selectivity fortransformed versus normal cells can be observed, as reflected in Table3. For example, compound example 1 (FIG. 3B) shows very high selectivityfor tumor cells as opposed to normal heart, brain or lung tissue. Theprecise mechanism for this selectivity is not known, although it isbelieved to arise from a higher affinity for tumor hsp90 as opposed tonormal cells hsp90.

The compositions of the invention are administered to subjects,including human patients, in need of treatment, in an amount effectiveto bring about the desired therapeutic result. A suitable administrationroute is intravenous administration, which is now commonly employed inchemotherapy. In addition, because the compositions of the inventionsare small soluble molecules, they are suitable for oral administration.The ability to use an oral route of administration is particularlydesirable where it may be necessary to provide treatment of a frequent,for example a daily schedule. The amount of any given composition to beadministered, and the appropriate schedule for administration aredetermined using toxicity tests and clinical trials of standard design,and will represent the conclusion drawn from a risk benefit analysis.Such analyses are routinely performed by persons skilled in the art, anddo not involve undue experimentation.

Due to the higher affinity these agents manifest towards cancer cellsand their preferential tumor retention profile, these agents are usefulas tumor imaging agents. They may also be used to monitor the responseof tumors to Hsp90-targeted therapy. The compounds of FIGS. 3F and G(Examples 8 and 9) were iodinated with ¹³¹I using chloramine T andtributyltin precursors. The radioligands were purified by Cl 8 RP HPLC.The saturation binding of these agents to CWR22-rvl prostate cancercells was undertaken. Animal biodistribution studies and MicroPETimaging were also performed in nude mice with CWR22 transplantabletumors using ¹²⁴I-labeled compounds. These studies showed the uptake ofthe radiolabeled compounds, and the ability to use these compounds inradioimaging to provide an image of a tumor.

Other options for radiolabeling include ¹⁸F which can be used inpositron emission tomography (PET). ¹²³I-labeled compounds can be usedin single photon emission computed tomography (SPECT), and ¹²⁵I-labeledcompounds can be used in surgical gamma probe detection.

The compositions of the invention also have utility to enhance thesensitivity of tumors to other forms of therapy, such as radiation andchemotherapy. This utility can be applied in the context of any type oftumor, but it is particularly relevant in the treatment of gliomas.Given the current therapeutic challenge due to radioresistance andchemoresistance explaining the poor prognosis (median survival of 12months) in GBM, identification of agents that may both sensitize gliomasto radiation and further act as treatments in inhibiting the growth ofthese tumors is necessary. Multipathway-targeted therapy using singleagents that target multiple pathways, including HDAC and Hsp90inhibitors hold promise for improved radiation therapy efficacy and,ultimately, improved patient outcome. Because radiotherapy remains aprimary treatment modality for gliomas, the ability to enhance gliomacell radiosensitivity should provide a therapeutic advantage. Previousstudies using 17 AAG and 17DMAG have suggested that Hsp90 is aclinically relevant target for the radiosensitization of a wide varietyof tumors (Russell et al, Clinical Cancer Research 9: 3749-3755, 2003;Bull et al, Clinical Cancer Research 10: 8077-8084, 2004). However,whereas in vitro studies have indicated that these Hsp90 inhibitorsenhance glioma cell radiosensitivity, 17AAG and 17DMAG do not penetratethe blood brain barrier and thus do not appear applicable to brain tumortherapy.

Compound 9 has been shown to have the ability to cross the blood brainbarrier and therefore is suitable for combination with radiotherapy as anovel form of brain tumor treatment. Initial studies based on theclonogenic survival assay indicate that Compound 9 enhances the in vitroradiosensitivity of two human glioma cell lines (U251 and U87) with doseenhancement factors of 1.4-1.6, a degree of radiosensitization similarto that previously shown for 17AAG and 17DMAG. Cells were exposed to 200nM or 400 nM Compound 8 or 9 for 16 h, irradiated with graded doses of Xrays, rinsed and fed with fresh growth media. Colony forming efficiencywas determined 10-12 days later and survival curves generated afternormalizing for cell killing by Compound 9 alone. The results aresummarized in Fig HA (Compound 9) and B (Compound 8).

The surviving fractions after Compound 9 treatment only were 0.86 and0.68 for U251 and U87 cells, respectively.

The following procedures and experiments were performed, and areprovided here to further demonstrate the invention.

Hsp90 competition assay. Fluorescence polarization measurements wereperformed on an ANALYST® AD instrument (Molecular Devices, Sunnyvale,Calif.). Measurements were taken in black 96-well microtiter plates(Corning #3650). The assay buffer (HFB) contained 20 mM HEPES (K) pH7.3, 50 mM KCl, 5 mM MgCl₂, 20 mM Na₂MoO₄, 0.01% NP40. Before each use,0.1 mg/mL bovine gamma globulin (BGG) (Panvera Corporation, Madison,Wis.) and 2 mM DTT (Fisher Biotech, Fair Lawn, N.J.) were freshly added.GM-BODIPY was synthesized as previously reported^(23a) and was dissolvedin DMSO to form 10 μM solutions. Recombinant Hsp90a was purchased fromStressgen Bioreagents (cat. No. SPP-776), (Victoria, Canada). Celllysates were prepared rupturing cellular membranes by freezing at -70°C. and dissolving the cellular extract in HFB with added protease andphosphotase inhibitors. Organs were harvested from a healthy mouse andhomogenized in HFB. Saturation curves were recorded in which GM-BODIPY(5 nM) was treated with increasing amounts of cellular lysates. Theamount of lysate that resulted in polarization (mP) readingscorresponding to 20 nM recombinant Hsp9060 was chosen for thecompetition study. For the competition studies, each 96-well contained 5nM fluorescent GM, cellular lysate (amounts as determined above andnormalized to total Hsp90 as determined by Western blot analysis usingas standard Hsp90 purified from HeLa cells (Stressgen#SPP-770) andtested inhibitor (initial stock in DMSO) in a final volume of 100 μL.The plate was left on a shaker at 4° C. for 7 h and the FP values in mPwere recorded. EC₅₀ values were determined as the competitorconcentrations at which 50% of the fluorescent GM was displaced.

Cell culture. The human breast cancer cell lines SKBr3 and MDA-MB-468were a gift from Dr. Neal Rosen (MSKCC). Cells were maintained in 1:1mixture of DME:F12 supplemented with 2 mM glutamine, 50 units/mLpenicillin, 50 units/mL streptomycin and 10% heat inactivated fetalbovine serum (Gemini Bioproducts#100-10b) and incubated at 37° C., 5%CO₂. Growth assays. Growth inhibition studies were performed using thesulforhodamine B assay as previously described.²⁹ In summary,experimental cultures were plated in microtiter plates (Nunc#167008).One column of wells was left without cells to serve as the blankcontrol. Cells were allowed to attach overnight. The following day,growth medium having either drug or DMSO at twice the desired initialconcentration was added to the plate in triplicate and was seriallydiluted at a 1:1 ratio in the microtiter plate. After 72 h of growth,the cell number in treated versus control wells was estimated aftertreatment with 50% trichloroacetic acid and staining with 0.4%sulforhodamine B in 1% acetic acid. The IC₅₀ was calculated as the drugconcentration that inhibits cell growth by 50% compared with controlgrowth. Normal human renal proximal tubular epithelial (RPTEC) cellswere purchased pre-seeded in 96-well plates (Clonetics, CC-3190). Uponreceipt, cells were placed in a humidified incubator at 37° C., 5% CO₂and allowed to equilibrate for 3 h. Media was removed by suction andreplaced with fresh media provided by the manufacturer. Cells were thentreated with either drugs or DMSO for 72 h and the IC₅₀ values weredetermined as described above.

Her2 assay. SKBr3 cells were plated in black, clear-bottom microtiterplates (Corning#3603) at 3,000 cells/well in growth medium (100 μl) andallowed to attach for 24 h at 37° C. and 5% CO2. Growth medium (100 μl)with drug or vehicle (DMSO) was carefully added to the wells, and theplates were placed at 37° C. and 5% CO₂. Following 24 h incubation withdrugs, wells were washed with ice-cold Tris buffer saline (TBS)containing 0.1% Tween 20 (TBST) (200 μl). A house vacuum source attachedto an eight-channel aspirator was used to remove the liquid from theplates. Further, methanol (100 μl at ˜20° C.) was added to each well,and the plate was placedat 4° C. for 10 min. Methanol was removed bywashing with TBST (2×200 μl). After further incubation at RT for 2 hwith SUPERBLOCK® R (Pierce 37535) (200 μl), anti-Her-2 (c-erbB-2)antibody (Zymed Laboratories#28-004) (1000, 1:200 in SUPERBLOCK® R) wasplaced in each well. The plate was incubated overnight at 4° C. Forcontrol wells, 1:200 dilution of a normal rabbit IgG (SantaCruz#SC-2027) in SUPERBLOCK® R was used. Each well was washed with TBST(2®μl) and incubated at RT for 2 h with an anti-rabbit HRP-linkedantibody (Sigma, A-0545) (100 μl, 1:2000 in SuperBlockR). Unreactedantibody was removed by washing with TBST (3×200 μl), and the ECLTMWestern blotting reagent (Amersham #RPN2106) (100 μL) was added. Theplate was immediately read in an Analyst AD plate reader (MolecularDevices). Each well was scanned for 0.1 s. Readings from wellscontaining only control IgG and the corresponding HRP-linked secondaryantibody were set as background and deducted from all measured values.

Luminescence readings resulted from drug-treated cells versus untreatedcells (vehicle treated) were quantified and plotted against drugconcentration to give the EC₅₀ values as the concentration of drug thatcaused 50% decrease in luminescence.

Anti-mitotic assay. Black, clear-bottom microtiter 96-well plates(Corning Costar#3603) were used to accommodate experimental cultures.MDA-MB-468 cells were seeded in each well at 8,000 cells per well ingrowth medium (100 μL), and allowed to attach overnight at 37° C. and 5%CO₂. Growth medium (100 μL) with drug or vehicle (DMSO) was gently addedto the wells, and the plates were incubated at 37° C. and 5% CO₂ for 24h. Wells were washed with ice-cold TBST (2×200 μL). A house vacuumsource attached to an eight-channel aspirator was used to remove theliquid from the 96-well plates. Ice-cold methanol (100 μL) was added toeach well, and the plate was placed at 4° C. for 5 min. Methanol wasremoved by suction and plates were washed with ice-cold TBST (2×200 μL).Plates were further incubated with SuperBlock® blocking buffer (Pierce#37535) (200 μL) for 2 h at RT. The Tg-3 antibody (gift of Dr. Davies,Albert Einstein College of Medicine) diluted 1:200 in SuperBlock® wasplaced in each well (100 μL) except the control column that was treatedwith control antibody (Mouse IgM, NeoMarkers, NC-1030-P). After 72 h,wells were washed with ice-cold TBST (2×200 μL). The secondary antibody(Goat Anti-Mouse IgM, SouthernBiotech #1020-05) was placed in each wellat 1:2000 dilution in SuperBlock® , and incubated on a shaker at RT for2 h. Un-reacted antibody was removed by washing the plates with ice-coldTBST (3×200 μL) for 5 min on a shaker. The ECLTM Western BlottingDetection Reagents 1 and 2 in 1:1 mix (100 μL) was placed in each welland the plates were read immediately in an Analyst AD plate reader(Molecular Devices). Luminescence readings were imported into SOFTmaxPROR 4.3.1. Anti-mitotic activity was defined as a concentrationdependent increase in luminescence readings in compound-treated wells ascompared to DMSO only treated wells.

General Chemical Procedures. AU commercial chemicals and solvents arereagent grade and were used without further purification. The identityand purity of each product was characterized by MS, HPLC, TLC, IR andNMR. ¹H NMR/¹³C NMR spectra were recorded on a Bruker 400 MHzinstrument. Low-resolution mass spectra (MS) were recorded in thepositive ion mode under electron-spray ionization (ESI). Highperformance liquid chromatography analyses were performed on a Waters2996 instrument with a photodiode array detector (read at 265 nm) and areverse-phase column (Higgins; HAISIL HL C18 5 μm) (method (a)) andadditionally, a Waters 2695 Separation Module with a Waters 996photodiode array detector and a Waters micromass ZQ and a reverse-phasecolumn (Varian; Microsorb 100-5 C18 150×2) (methods (b) and (c)). Method(a): 0.1% TFA in water-acetonitrile in the indicated ratio; method (b):0.05% TFA in water-0.04% TFA in acetonitrile; method (c): 0.05% TFA inwater-0.04% TFA in acetonitrile gradient (35% acetonitrile over 18 min,35-95% acetonitrile over 6 min, 95% acetonitrile over 9 min). Infraredspectra (IR) were obtained on a Perkin-Elmer FT-IR model 1600spectrometer. Characterization data for previously unknown compoundswere determined from a single run with isolated yields. Reactions weremonitored by thin-layer chromatography on 0.25-mm silica gel plates andvisualized with UV light. Column chromatography was performed usingsilica gel (Fisher 170-400 mesh) or alumina (Fisher 60-325 mesh).Oxidation reactions with OXONE® were carried out in the presence of theFisher alumina (A540; 80-200 mesh). Analytical thin-layer chromatography(TLC) was performed on E. Merck precoated silica gel 60 F254. WatersSep-PakR Vac 6cc (500 mg) Cl 8 cartridges were used for the purificationof compounds 16. All reactions were conducted under inert atmosphereexcept of those in aqueous media.

3,4,5-Trimethoxy-benzenethiol (6). To 3,4,5-trimethoxyaniline (2 g, 10.9mmol) at 0° C. were added a concentrated solution of HCl (3 mL, 0.27mL/mmol) and H₂O (7.7 mL) followed by NaNO₂ (932 mg, 13.1 mmol). Theresulting solution was poured over potassium ethyl xanthogenate (5.35 g,32.7 mmol) in H₂O (6.2 mL) and stirred at 50° C. for 40 min The reactionmixture was brought to room temperature, diluted with EtOAc (80 mL) andwashed with 10% NaOH, followed by H₂ O until the pH reached 7. Theorganic fraction was dried over Na₂SO₄ and the solvent evaporated underhigh vacuum. The residue was purified by column chromatography on silicagel (CH₂Cl₂) to furnish the xanthogenate intermediate (1.82 g, 58%yield). This was taken up in anhydrous THF (30 mL). To the resultingsolution, LiAlH₄ (I g, 25 mmol) was slowly added and the mixture wasstirred for 1 h at reflux temperature. Following cooling to roomtemperature, the reaction was quenched with ice cold water (50 mL) and10% H₂SO₄ (5 mL) and extracted with CHCl₃. The organic phase was driedover Na₂SO₄ and evaporated to give the desired thiophenol (1.18 g, 93%yield). ¹H NMR (CDCl₃) δ6.53 (s, 2H), 3.84 (s, 6H, OCH3), 3.82 (s, 3H,OCH3), 3.46 (s, ¹H, SH).

Procedures for the formation of 8-arylsulfanyladenine derivatives:Scheme 1, synthetic step (b). 8-Mercaptoadenine (7) (50.2 mg, 0.30mmol), neocuproine hydrate (6.8 mg, 0.03 mmol), CuI (5.7 mg, 0.03 mmol),NaO-t-Bu (57.6 mg, 0.6 mmol), the corresponding aryl iodide (0.90 mmol)and anhydrous DMF (2 mL) were charged in a nitrogen box. The reactionvessels were sealed with Teflon tape, placed in an oil bath (110° C.)and magnetically stirred for 24 h. The reaction mixture was then cooledto room temperature and DMF was removed in vacuo. The crude was purifiedby silica gel flash chromatography eluting with a gradient ofCHCl₃:NH₄OH at 10:0.5 to CHCl₃ :MeOH:NH₄OH at 10:1:0.5 to afford thedesired product.

8-(2,4,5-Trichloro-phenyIsulfanyl)adenine. Use K₂CO₃ as base. Yield,56%. ¹H NMR (400 MHz, DMSO-d6) δ8.12 (s,1H), 8.00 (s,1H), 7.60 (s,1H),7.37 (s, 2H); ¹³C NMR (100 MHz, DMSO-d6) δ132.5, 132.3, 132.2, 131.4,131.2, 130.8; MS m/z 345.9 (M+H)+. HPLC: (a) 99.9% (65% water-35%acetonitrile); (b) 99.4%.

Scheme 1, synthetic step (d): A mixture of 8-arylsulfanyl adenine 10(100 μmol), Cs2CO3 (100 μmol), pent-4-ynyl 4-methylbenzenesulfonate (120μmol) in DMF (1.3 mL) under nitrogen protection was heated at 80° C. for30 min Following solvent removal, the crude was purified by preparatoryTLC with CHCl₃:MeOH:NH4OH at 10:1:0.5 or CHCl₃ :MeOH: AcOH at 10:1:0.5to provide the corresponding 9-alkyl-8-arylsulfanyladenine derivatives11.

9-(Pent-4-ynyl)-8-(2,4,5-trichloro-phenyIsulfanyI)adenine (Hu). Yield,46%. ¹H NMR (400 MHz, CDC13/MeOD-d4) δ8.26 (s,1H), 7.63 (s,1H), 7.50(s,1H), 4.38-4.35 (t, J=7.3 Hz, 2H), 2.32-2.28 (m, 2H), 2.09-2.02 (m,3H); ¹³C NMR (100 MHz, CDC13/MeOD-d4) 67 154.4, 152.7, 150.9, 143.8,134.1, 133.7, 133.4, 131.9, 131.3, 129.4, 81.8, 69.4, 42.9, 28.1, 15.7;MS m/z 411.9 (M+H)+. HPLC: (a) 98.5% (75% water-25% acetonitrile) ; (b)97.1%.

9-(Pent-4-ynyl)-8-(6-bromo-benzo[1,3]dioxol-5-ylsulfanyl)adenine. Yield,48%. ¹H NMR (400 MHz, CDC13/MeOD-d4) δ8.22 (s,1H), 7.17 (s,1H), 7.00(s,1H), 6.06 (s, 2H), 4.35-4.31 (t, J=7.26 Hz, 2H), 4.12 (s, 2H),2.33-2.30 (m, 2H), 2.08-2.05 (m, 3H). ¹³C NMR (100 MHz, CDC13/MeODd4)δ150.9, 149.7, 148.0, 146.9, 121.3, 119.1, 113.8, 113.4, 102.4, 81.9,69.3, 42.7, 28.0, 15.6; MS m/z 432.0 (M+H)+. HPLC: (a) 98.7% (75%water-25% acetonitrile); (b) 98.9%.

9-(2-Isopropoxy-ethyl)-8-(2,4-dichloro-5-methoxy-benzenesulfanyl)adenine.Following the general method for the preparation of 12d, Ile3 afforded12c. Yield, 53%. ¹H NMR (400 MHz, CDC13) δ8.36 (s,¹H, H-2), 7.44 (s,1H),7.09 (s,1H), 5.55 (bs, 2H, NH2), 4.48 (t, J=5.6 Hz, 2H, NCH2), 3.81 (s,3H, OCH3), 3.74 (t, J=5.6 Hz, 2H), 3.49 (d, J=6.1 Hz, ¹H, CH), 1.04 (d,J=6.1 Hz, 6H, CH3); 13C NMR (100 MHz, CDC13) δ154.1 (C-2), 153, 147,131.0, 130.2, 126.9, 123.6, 116.0, 72.3 (CH), 65.6, 56.5 (OCH3), 44.1(NCH2), 21.8 (CH2); MS (EIS) m/z 428.0 (M+1)+. HPLC: (a) 90.2% (70%water-30% acetonitrile); (c) 91.0%.

Method for the fluorination of the adenine moiety at C2:2-Fluoro-9-butyl- 8-(2-chloro-3,4,5-trimethoxy-phenylsulfanyl)adenine(21c). To a cooled solution (0° C.) of 18c (11.3 mg, 0.02 mmol) inHF/pyridine (18 μL, 0.7 mL/mmol) NaNO2 (2.2 mg, 0.03 mmol) was slowlyadded. The resulting mixture was stirred at room temperature for 1 h andthen quenched by stirring for 1 h with 14 mg of CaCCβ in CH2C12 (75 μL).The crude was taken up in CH2C12, washed with water and dried overanhydrous Na2SO4. Following solvent removal, the residue was purified ona preparative silica gel plate (CHC13:Hexanes:EtOAc:i-PrOH at 2:2:1:0.1)to afford 21c (1.9 mg, 17% yield). IR (film) v_(max) 3318-2953, 1657,1604, 1583, 1479, 1385, 1111, 1015; ¹H NMR (400 MHz, CDC13) δ6.72(s,1H), 5.83 (bs, 2H, NH2), 4.18 (t, J=7.5 Hz, 2H, NCH2), 3.92 (s, 3H,OCH3), 3.89 (s, 3H, CH3), 3.74 (s, 3H, CH3), 1.72 (m, 2H), 1.32 (m, 2H),0.92 (t, J=7.4 Hz, 3H, CH3); ¹³C NMR (100 MHz, CDC13) δ160.1, 158.0,156.1, 152.5, 150.8, 143.9, 124.6, 111.2, 61.2 and 56.3 (OCH3), 43.9(NCH2), 31.7, 29.7, 19.7 (CH3); MS (EIS) m/z 442.2 (M+1)+. HPLC: (a)95.9% (60% water-40% acetonitrile); (c) 98.0%.

8-(6-Bromo-benzo[1,3]dioxol-5-ylsulfanyI)adenine. 8-Mercaptoadenine (602mg, 3.6 mmol), neocuproine hydrate (81 mg, 0.36 mmol), CuI (69 mg, 0.36mmol), NaO-t-Bu (692 mg, 7.2 mmol), 5-bromo-6-iodo˜benzo[1,3]dioxole(3.53 g, 10.8 mmol) and anhydrous DMF (24 mL) were charged in a nitrogenbox. The vessel was sealed with Teflon tape, placed in an oil bath (110°C.) and magnetically stirred for 24 h. The solvent was removed underhigh vacuum and the crude purified by column chromatography on silicagel (EtOAc:CH2C12:MeOH at 2:2:1) to provide the product (1.29 g, 97%).¹H NMR (400 MHz, acetone-d6) 8.07 (s,1H), 7.28 (s,1H), 7.15 (s,1H), 7.08(bs, 2H), 6.13 (s, 2H); MS m/z 366.0 (M+H)+.

8-(6-Iodo-benzo[1,3]dioxol-5-ylsulfanyl)-9-(pent-4-ynyI)adenine: Asolution of 7 (40 mg, 96.8 mol), Cs2CO3 (31.5 mg, 96.8 mol) andpent-4-ynyl tosylate (28 mg, 114 mol) in anhydrous DMF (1 mL) wasstirred at 80° C. for 30 min. The solvent was removed under high vacuumand the crude purified by preparatory thin layer chromatography to givethe desired product (25 mg, 53.9%): ¹H NMR (400 MHz, CDC13/methanol-d4)8.23 (s,1H), 7.38 (s,1H), 7.04 (s,1H), 6.05 (s, 2H), 4.32 (t, J=7.3 Hz,2H), 2.33-2.31 (m, 2H), 2.12-2.04 (m, 3H); 13C NMR (100 MHz,CDC13/methanol-d4) 151.1, 149.6, 149.2, 147.5, 125.7, 119.4, 113.6,102.4, 93.8, 82.1, 69.4, 42.8, 28.1, 15.8; MS m/z 480.0 (M+H)+. HPLC:(a) 98.5% (65% water-35% acetonitrile); (b) 97.7% (35% to 95%acetonitrile).

3-(tert-Butoxycarbonyl-isopropyl-amino)-propyl tosylate: A solution of3-bromo-1-propanol (5g, 0.036 mol) in isopropylamine (9 mL, 0.11 mol)was heated overnight at 50° C. with stirring. Solvent was removed undervacuum to give the product, 3-isopropyl-amino-propanol as a white solid.To this were added di-tert-butyl dicarbonate (10 g, 0.05 mol) andtriethylamine (11 mL, 0.08 mol) and the resulting solution stirred atroom temperature overnight. Following solvent removal, the crude waspurified by column chromatography on silica gel (CH2C12, then CH2C12:acetone at 3:1) to provide the3-(tert-butoxycarbonyl-isopropyl-amino)-propanol (5.8 g, 75%). ¹H NMR(400 MHz, CDC13) 3.93 (bs,1H), 3.58 (m, 2H), 3.33 (m, 2H), 1.67 (m, 2H),1.48 (s, 9H), 1.16 (d, J=6.9 Hz, 6H); MS m/z 218.1 (M+H)+. A solution of

3-(tert-butoxycarbonyl-isopropyl-amino)-propanol (3.5 g, 0.016 mol),p-toluenesulfonyl chloride (3.7 g, 0.019 mol) and pyridine (1.6 mL,0.019 mol) in CH2C12 (50 mL) was stirred overnight at room temparature.Following solvent removal, the product (2.3 g, 40%) was isolated bycolumn chromatography on silica gel (hexanes:CH2C12:EtOAc at 5:4:1). ¹HNMR (400 MHz, CDC13) 7.79 (d, J=8.2 Hz, 2H,), 7.35 (d, J=8.2 Hz, 2H,),4.06-4.03 (m, 3H), 3.09 (t, J=6.5 Hz, 2H), 2.45 (s, 3H), 1.91-1.87 (m,2H), 1.42 (s, 9H), 1.08 (d, J =6.7 Hz, 6H); MS m/z 372.2 (M+H)+.

8-(6-Iodo-benzo[1,3]dioxol-5-yIsulfanyl)-9-(3-isopropylamino-propyl)adenine:A solution of 7 (125 mg, 303 mol),3-(tert-butoxycarbonyl-isopropyl-amino)-propyl tosylate (269 mg, 726mol), Cs2CO3 (99 mg, 303 mol) in anhydrous DMF (2 mL) was stirred at 80°C. for 24 h. The solvent was removed under vacuum and the crude purifiedby preparatory thin layer chromatography on silica gel (CHC13:MeOH:NH4OHat 10:1:0.5) to afford the 9N-alkylated compound. This was placed in TFA(1 mL) at 0° C. for 1.5 h to remove the Boc protecting group and yield11 (30 mg, 19.3% yield): ¹H NMR (400 MHz, CDC13) 8.31 (s,1H), 7.29(s,1H), 6.88 (s,1H), 6.10 (bs, 2H), 5.96 (s, 2H), 4.29 (t, J=7.0 Hz,2H), 2.75-2.69 (m,1H), 2.58 (t, J=6.8 Hz, 2H), 2.02-1.95 (m, 2H), 1.03(d, J=6.2 Hz, 6H); 13C NMR (100 MHz, CDC13) 154.6, 152.9, 151.6, 149.2,148.9, 146.2, 127.9, 120.1, 119.2, 112.2, 102.2, 91.1, 48.7, 43.9, 41.7,30.3, 22.9; MS m/z 513.2 (M+H)+. HPLC: (a) 98.9% (65% water-35%acetonitrile); (b) 95.0% (20% to 40% acetonitrile).

8-(6-Bromo-benzo[1,3]dioxol-5-yIsulfanyl)-9-(3-isopropylamino-propyl)adenine:A solution of 8-(6-Bromo-benzo[1,3]dioxol-5-ylsulfanyl)adenine (513 mg,1.4 mmol), PPh3 (808 mg, 3.08 mmol), 3-bromo-1-propanol (253 mg, 165 L,1.82 mmol), DBAD (1612 mg, 7 mmol) in toluene (43.8 mL) and CH2C12(8.75, mL) was stirred at room temperature for 20 min. The reactionmixture was loaded to a silica gel column (CHC1₃ thenCHC1₃:EtOAc:hexanes: i-Propanol at 4:4:2:1) to provide the 9N-alkylatedcompound,8-(6-bromo-benzo[1,3]dioxol-5-ylsulfanyl)-9-(3-bromo-propyl)adenine)(142.6 mg, 21% yield). A solution of this product (142.6 mg, 0.29 mmol)in 1,4-dioxane (12 mL) and i-propylamine (3 mL) was stirred at 100° C.for 2.5 h. The solvent was removed under vacuum and the crude purifiedby preparatory thin layer chromatography on silica gel (CHC13:MeOH:NH4OHat 10:1:0.5 then EtOAc:CH2C12:MeOH at 2:2:1) to afford 12 (51 mg, 8%yield). ¹H NMR (400 MHz, CDC13) 8.30 (s,1H), 7.04 (s,1H), 6.81 (s,1 H),6.48 (bs, 2H), 5.94 (s, 2H), 4.29 (t, J=7.0 Hz, 2H,), 2.74-2.68 (m,1H),2.57 (t, J=6.8 Hz, 2H,), 2.02-1.95 (m, 2H), 1.02 (d, J=6.0 Hz, 6H). 13CNMR (100 MHz, CDC13) 154.8, 152.9, 151.5, 148.8, 148.0, 145.2, 123.8,120.0, 116.7, 113.2, 112.2, 102.3, 48.6, 43.8, 41.7, 30.2, 22.8; MS m/z465.0 (M+H)+. HPLC: (a) 99.1% (65% water-35% acetonitrile); (b) 98.0%(20% to 40% acetonitrile).

8-Benzo[1,3]dioxol-5-ylmethyl-2-fluoroadenine: To a cooled (0° C.)solution of 16 (1.48 g, 5.2 mmol) in HF/pyridine (3.64 mL), NaNO2 (0.47g, 6.76 mmol) was slowly added. The reaction was brought to roomtemperature, and further stirred for 1 h. Following dilution with CH₂Cl₂(38 mL), the excess HF was quenched by stirring for an additional 1 hwith CaCO3 (0.95 g) and water (5 mL). The mixture was dried in vacuo andsubsequently purified by silica gel column chromatography(CHC13:MeOH:NH4OH at 5:1:0.5) to yield 17 (0.9 g, 60% yield). ¹H NMR(400 MHz, DMSO-d6) 7.59 (bs, 2H), 6.94-6.90 (m, 3H), 6.81 (d, J=8.0Hz,1H), 6.03 (s, 2H), 4.06 (s, 2H); MS m/z 288.0 (M+H)+.

2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)adenine: A solution of8-Benzo[1,3]dioxol-5-ylmethyl-2-fiuoroadenine (50 mg, 0.17 mmol), NIS(94 mg, 0.4 mmol), TFA (20 mg, 13.4 L, 0.17 mmol) in CH2C12 (200 L) wasstirred at room temperature overnight. After solvent removal, thedesired product 18 (6 mg, 8.5%) was purified by silica gel columnchromatography (CHC13:EtOAc at 9:1 to 4:6). ¹H NMR (400 MHz, DMSO-d6)7.6 (bs, 2H), 7.38 (s,1H), 6.95 (s,1H), 6.03 (s, 2H), 4.12 (s, 2H); MSm/z 414.1 (M+H)+.

2-Fluoro-8-(6-bromo-benzo[1,3]dioxol-5-ylmethyl)adenine: A solution of8-Benzo[1,3]dioxol-5-ylmethyl-2-fiuoroadenine (45 mg, 0.157 mmol), NBS(56 mg, 0.314 mmol) in DMF (0.5 mL) was stirred at room temperature for1.5 h. Following solvent removal, product (20 mg, 34.8%) was collectedthrough silica gel column purification (CHC13:EtOAc at 9:1 to 4:6). ¹HNMR (400 MHz, acetone-d6) 7.51 (bs, 2H), 7.21 (s,1H), 6.98 (s,1H), 6.06(s, 2H), 4.13 (s, 2H); MS m/z 366.0 (M+H)+.

2-Fluoro-8-(6-chloro-benzo[1,3]dioxol-5-yImethyI)adenine: A solution of8-Benzo[1,3]dioxol-5-ylmethyl-2-fluoroadenine (20 mg, 0.07 mmol), NCS(35.6 mg, 0.27 mmol) in anhydrous DMF (0.4 mL) was stirred at roomtemperature for 2.5 h. Following solvent removal, the product (11 mg,48.8%) was collected through silica gel column purification (CHC13:EtOAcat 9:1 to 5:5). ¹H NMR (400 MHz, DMSO-d6) 7.40 (bs, 2H), 6.97 (s,1H),6.89 (s, 2H), 5.97 (s, 2H), 4.04 (s, 2H); MS m/z 322.1 (M+H)+.

2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-yïmethyl)-9-(pent-4-ynyl)adenine:A solution of 2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)adenine (6mg, 0.0145 mmol), Cs2CO3 (5 mg, 0.0145 mmol) and pent-4-ynyl tosylate(4.5 mg, 0.189 mmol) in anhydrous DMF (200 L) was stirred at 60° C. for1.5 h. Following solvent removal, product (5.9 mg, 84.9%) was collectedthrough silica gel column purification (EtOAc:hexanes:CHC13:i-PrOH at10:20:20:1). ¹H NMR (400 MHz, CDC13) 7.29 (s,1H), 6.59 (s,1H), 5.94 (s,2H), 5.83 (bs, 2H), 4.26 (s, 2H), 4.11 (t, J=7.4 Hz, 2H), 2.26-2.19 (m,2H), 2.00 (t, J=2.5 Hz,1H), 1.98-1.94 (m, 2H); 13C NMR (IOO MHZ, CDC13)150.9, 148.9, 147.8, 131.5, 118.8, 109.4, 101.9, 88.1, 82.3, 69.9, 42.3,39.2, 28.2, 15.9; MS m/z 480.0 (M+H)+. HPLC: (a) 95.5% (60% water-40%acetonitrile); (b) 95.0% (35% to 55% acetonitrile).

2-Fluoro-8-(6-bromo-benzo[1,3]dioxol-5-ylmethyl)-9-(pent-4-ynyl)adenine:A solution of 2-Fluoro-8-(6-bromo-benzo[1,3]dioxol-5-ylmethyl)adenine(20 mg, 55 mol), Cs2CO3 (18 mg, 55 mol) and pent-4-ynyl tosylate (17 mg,72 mol) in anhydrous DMF (138 L) was stirred at 60° C. for 2 h.Following solvent removal, the product (13 mg, 54.7%) was collectedthrough silica gel column purification (EtOAc:hexanes:CHC13:i˜PrOH at10:20:20:1). ¹H NMR (400 MHz, CDC13) 7.05 (s,1H), 6.60 (s,1H), 6.15 (bs,2H), 5.96 (s, 2H), 4.28 (s, 2H), 4.13 (t, J=7.5 Hz, 2H), 2.25-2.21 (m,2H), 2.00 (t, J=2.6 Hz,1H), 1.98-1.92 (m, 2H); 13C NMR (100 MHz, CDC13)157.6, 156.0, 152.6, 150.2, 147.5, 127.6, 116.4, 114.1, 112.5, 109.5,101.6, 81.9, 69.5, 41.9, 33.7, 27.8, 15.5; MS m/z 432.0 (M+H)+. HPLC:(a) 99.0% (60% water-40% acetonitrile); (b) 98.5% (35% to 55%acetonitrile).

2-Huoro-8-(6-chloro-benzo[1,3]dioxoI-5-ylmethyI)-9-(pent-4-ynyl)adenine:A solution of 2-Fluoro-8-(6-chloro-benzo[1,3]dioxol-5-ylmethyl)adenine(11 mg, 0.034 mmol), Cs2CO3 (11 mg, 0.034 mmol) and pent-4-ynyl tosylate(10.5 mg, 0.044 mmol) in anhydrous DMF (85 L) was stirred at 50° C. for1 h. Following solvent removal, the product (4.2 mg, 31.9%) wascollected through silica gel column purification(EtOAc:hexanes:CHC13:i-PrOH at 10:20:20:1). ¹H NMR (400 MHz, CDC13) 6.89(s,1H), 6.61 (s,1H), 5.98 (bs, 2H), 5.96 (s, 2H), 4.27 (s, 2H), 4.13 (t,J=7.5 Hz, 2H), 2.24-2.10 (m, 2H), 2.00-1.91 (m, 3H); 13C NMR (100 MHz,CDC13) 1597, 158.0, 156.3, 150.6, 147.7, 147.2, 126.2, 125.3, 110.0,102.0, 82.3, 69.9, 42.2, 31.4, 28.1, 15.8; MS m/z 388.1 (M+H)+. HPLC:(a) 98.1% (65% water-35% acetonitrile); (b) 97.0% (35% to 45%acetonitrile).

2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyI)-9-(3-isopropyIamino-propyI)adenine: A solution of2-Fluoro-8-(6-iodo-benzo[1,3]dioxol-5-ylmethyl)adenine (300 mg, 0.726mmol), Cs2CO3 (285 mg, 0.87 mmol) and 1,3-dibromopropane (370 L, 3.63mmol) in anhydrous DMF (5 mL) was stirred at 50° C. for 2 h. Followingsolvent removal, product (330 mg, 85%) was collected through silica gelcolumn purification (CHC13 then EtOAc:hexanes:CHC13:i-PrOH at4:2:4:0.4). MS m/z 534.0 (M+H)+. To this product, i-PrNH2 (10 mL) wasadded in excess and the resulting solution stirred at room temperaturefor 1 h. Excess amine was removed and product (230 mg, 75%) collectedthrough silica gel column purification (CHC13:EtOAc:i-PrOH:NH4OH at4:4:2:0.3). ¹H NMR (400 MHz, CDC13) 7.29 (s,1H), 6.59 (s,1H), 5.94 (s,2H), 5.89 (bs, 2H), 4.25 (s, 2H), 4.11 (t, J=7.0 Hz, 2H), 2.73-2.60(m,1H), 2.55 (t, J=6.8 Hz,1H), 1.93-1.86 (m, 2H), 1.03-1.02 (d, J=6.0Hz, 6H); 13C NMR (100 MHz, methanol-d4) 160.0, 158.4, 157.2, 152.4,151.3, 149.4, 148.4, 133.1, 118.7, 110.6, 102.4, 88.5, 42.8, 40.1, 38.8,27.6, 19.4; MS m/z 513.2 (M+H)+. HPLC: (a) 98.5% (60% water-40%acetonitrile); (b) 97.2% (20% to 50% acetonitrile).

2-Fluoro-8-(3,4-dimethoxy-benzyl)adenine: Starting from2-amino-8-(3,4-dimethoxy-benzyl) adenine (0.66 g, 2.2 mmol) andfollowing the procedure for the synthesis of 17, the desired product wasobtained (0.34 g, 51%). ¹H NMR (400 MHz, DMSO-d6) 7.61 (bs, 2H), 7.03(s,1H), 6.94 (d, J =8.3 Hz,1H), 6.84 (d, J=8.6 Hz,1H), 4.07 (s, 2H),3.80 (s, 3H), 3.77 (s, 3H); MS m/z 304.0 (M+H)+.

2-Fluoro-8-(2-iodo-4,5-dimethoxy-benzyl)adenine: A solution of2-Fluoro-8-(3,4-dimethoxy-benzyl)adenine (50 mg, 0.165 mmol), NIS (74mg, 0.33 mmol), TFA (18.8 mg, 12.7 L, 0.165 mmol) in acetonitrile (120L) was stirred at room temperature for 24 h. Following solvent removal,the product (12 mg, 16.9%) was collected through silica gel columnpurification (CHC13:MeOH:AcOH at 80:1:0.5 to 30:1:0.5). MS m/z 430.1(M+H)+.

2-Fluoro-8-(2-bromo-4,5-dimethoxy-benzyl)adenine (29): A solution of 27(65 mg, 0.226 mmol), NBS (80 mg, 0.45 mmol) in DMF (0.75 mL) was stirredat room temperature for 2.5 h. Following solvent removal, the product(8.2 mg, 53.6%) was collected through silica gel column purification(CHC13:MeOH:AcOH at 80:1:0.5 to 30:1:0.5). ¹H NMR (400 MHz, aceton-d6)7.13 (s,1H), 7.09 (s,1H), 6.80 (bs, 2H), 4.26 (s, 2H), 3.84 (s, 3H),3.78 (s, 3H); MS m/z 382.0 (M+H)+.

2-Fluoro-8-(2-chloro-4,5-dimethoxy-benzyI)adenine: A solution of2-Fluoro-8-(3,4-dimethoxy-benzyl)adenine (40 mg, 0.132 mmol), NCS (77.8mg, 0.58 mmol) in anhydrous DMF (0.7 mL) was stirred at room temperaturefor 5.5 h. Following solvent removal, the product (22 mg, 49.4%) wascollected through silica gel column purification (CHC13:EtOAc at 8:2 to4:6). MS m/z 338.0 (M+H)+.

2-Fluoro-8-(2-iodo-4,5-dimethoxy-benzyl)-9-(pent-4-ynyl)adenine: Asolution of 2-fluoro-8-(2-iodo-4,5-dimethoxy-benzyl)adenine (12 mg,0.028 mmol), Cs2CO3 (9 mg, 0.028 mmol), pent-4-ynyl tosylate (8.6 mg, 7L, 0.036 mmol) in anhydrous DMF (80 L) was stirred at 50° C. for 1 h.Following solvent removal, the product (13.7 mg, 99%) was collectedthrough silica gel column purification (CHC13:EtOAc:hexanes:i-PrOH at20:10:20:1). ¹H NMR (400 MHz, CDC13) 7.27 (s,1H), 6.65 (s,1H), 5.94 (bs,2H), 4.29 (s, 2H), 4.13 (t, J=7.3 Hz, 2H), 3.87 (s, 3H), 3.73 (s, 3H),2.26-2.22 (m, 2H), 2.00 (t, J=2.6 Hz,1H), 1.97-1.90 (m, 2H); 13C NMR(100 MHz, CDC13) 156.5, 153.2, 151.3, 150.0, 149.1, 130.9, 121.9, 112.6,88.5, 82.5, 70.0, 56.4, 56.2, 42.6, 39.2, 28.4, 16.1; MS m/z 496.2(M+H)+. HPLC: (a) 99.9% (60% water-40% acetonitrile); (b) 96.8% (35% to55% acetonitrile).

2-Fluoro-8-(2-bromo-4,5-dimethoxy-benzyl)-9-(pent-4-ynyI)adenine: Asolution of 8-(2-bromo-4,5-dimethoxy-benzyl)-2-fluoroadenine (13 mg,0.034 mmol), Cs2CO3 (11 mg, 0.034 mmol), pent-4-ynyl tosylate (10 mg, 9L, 0.044 mmol) in anhydrous DMF (80 L) was stirred at 60° C. for 30 min.Following solvent removal, the product (8.2 mg, 53.6%) was collectedthrough silica gel column purification (CHC13:EtOAc:hexanes:i-PrOH at20:10:20:1). ¹HNMR (400 MHz, CDC13) 7.06 (s,1H), 6.67 (s,1H), 5.92 (bs,2H), 4.31 (s, 2H), 4.14 (t, J =7.4 Hz, 2H), 3.88 (s, 3H), 3.75 (s, 3H),2.25-2.20 (m, 2H), 1.99 (t, J=2.6 Hz,1H), 1.96-1.89 (m, 2H); 13C NMR(100 MHz, CDC13) 160.0, 158.3, 156.7, 153.4, 151.3, 149.4, 127.2, 117.3,115.9, 114.5, 113.2, 82.7, 70.2, 56.62, 56.56, 42.7, 34.3, 30.1, 28.5,16.3; MS m/z 447.9 (M+H)+. HPLC: (a) 99.0% (60% water-40% acetonitrile);(b) 98.8% (35% to 55% acetonitrile).

2-Fluoro-8-(2-chloro-4,5-dimethoxy-benzyl)-9-(pent-4-ynyI)adenine: Asolution of 8-(2-chloro-4,5-dimethoxy-benzyl)-2-fluoroadenine (22 mg,0.065 mmol), Cs2CO3 (21 mg, 0.065 mmol), pent-4-ynyl tosylate (20 mg,17.3 L, 0.085 mmol) in anhydrous DMF (170 L) was stirred at 50° C. for 2h. Following solvent removal, the product (14 mg, 53.8%) was collectedthrough silica gel column purification (CHC13:Et0Ac:hexanes:i-PrOH at20:10:20:1). ¹H NMR (400 MHz, CDC13) 6.91 (s,1H), 6.67 (s,1H), 6.01 (bs,2H), 4.31 (s, 2H), 4.14 (t, J=7.5 Hz, 2H), 3.87 (s, 3H), 3.75 (s, 3H),2.24-2.20 (m, 2H), 2.01-1.99 (m,1H), 1.97-1.88 (m, 2H); 13C NMR (100MHz, CDC13) 1596, 157.9, 156.3, 152.3, 148.9, 146.0, 124.9, 112.7, 82.3,69.9, 56.3, 56.2, 42.2, 31.2, 28.1, 15.9; MS m/z 404.1 (M+H)+. HPLC: (a)95.1% (65% water-35% acetonitrile); (b) 96.7% (35% to 45% acetonitrile).

Radiolabeling of Examples 8 and 9

Ten microliters of [¹³¹I]-NaI (3mCi) in 0.1 M NaOH is added to a 0.3 mLReactiVial followed by 5 μL of a 5 μg/ptL methanol solution of2-fluoro-9-[3-(N-N-tert-butoxycarboxy- 2-propylamino)propyl]-8-(4-trimethylstannyl-1,3-benzodioxol -5-yl)methyl adeninefollowed by 10 μL of Chloramine-T (CAT) in acetic acid (0.5 mg/mL). Thereaction mixture is vortexed and kept at 50° C. for 5 minutes. 10 μL 6MHCl is added, the reaction mixture is vortexed and kept at 50° C. for 15minutes. 6/xL 1 OM NaOH is added, the reaction mixture is vortexed andinjected into a HPLC (Phenomenex Luna Cl 8 column (5 μm, 4.4×250 mm)Both columns were eluted at 1 mL/min with a solvent gradient of 0.1% TFAto 0.1% TF A/70% acetonitrile over 15 minutes. The PU-DZ8 fraction iscollected, dried at 50° C. by a stream of nitrogen, reconstituted insaline and sterile filtered to yield -90% radiochemical yield of[¹³¹I]-Compound 8. [¹²⁴I]-Compound 8 and [¹³¹I]-Compound 9 are producedin an analogous manner.

Binding studies

CWR22-rvl prostate cancer cells are grown in RMPI 1640 mediasupplemented with 10% fetal bovine serum at 37° C. The cells are removedfrom the flasks using trypsin and propagated with a 1:6 subcultureratio.

Displacement Binding Studies

Displacement studies are performed with [¹³¹I]-Compound 8 and CWR22-rvlprostate cancer cells. Briefly, triplicate samples of cells are mixedwith <1 nM of radioligand and increasing amounts of a cold competitor (1pM to 1 μM Compound 8 or 9). The solutions are shaken on an orbitalshaker and after 60 minutes the cells are isolated and washed with icecold Tris buffered saline using a Brandel cell harvester. AU theisolated cell samples are counted and the specific uptake. of[¹³¹I]-Compound 8 determined. These data are plotted against theconcentration of the cold competitor to give sigmoidal displacementcurves. The IC₅₀ values are determined using a one site model and aleast squares curve fitting routine. The displacement binding of[¹³¹I]-Compound 8 is determined in an analogous manner FIG. 11 showsexample displacement curves for and [¹³¹I]-Compound 8 and[¹³¹I]-Compound 9.

Saturation Binding Studies

Saturation studies are performed with [¹³¹I]-Compound 8 and CWR22-rvlprostate cancer cells. Briefly, triplicate samples of cells are mixedwith increasing amount of 131I-DZ8 either with or without 1 μM unlabeledCompound 9. The solutions are shaken on an orbital shaker and after 60minutes the cells are isolated and washed with ice cold Tris bufferedsaline using a Brandel cell harvester. AU the isolated cell samples arecounted and the specific uptake of ¹³¹I-Compound 8 determined. Thesedata are plotted against the concentration of ¹³¹I-Compound to give asaturation binding curve. The Bmax (maximal binding) and Kd (bindingaffinity) are determined by using a least squares curve fitting routine.The saturation binding of [¹³¹I]-Compound 9 is determined in ananalogous manner. FIG. 12 shows example saturation curves for[¹³¹]-Compound 8 and [¹³¹I]-Compound 9.

Animal Studies

[¹³¹I]-Compound 8 and [¹³¹I]-Compound 9 biodistribution was studied inan animal model of prostate cancer. CWR22 tumors are grown in athymicmice supplemented with a testosterone pellet (12.5 mg pellet, InnovativeResearch of America, Sarasota, Fla.). Once the tumors are 500 mg in sizethe pellet is removed and the mice castrated.

Two groups of eight mice were injected at 3 days post castration, with10 μCi of [¹³¹I]-Compound 8 or [¹³¹I]-Compound 9. These mice weresacrificed at either 4 or 24 hours post injection and the organs ofinterest removed, weighed and counted in a gamma counter with a standardof 10% of the [¹³¹I]-Compound 8 injected dose. The data were thenexpressed as a % of the injected dose per gram of tissue (% ID/g). FIG.13 shows the tumor/non-tumor uptake ratios for selected organs. Asshown, there is a substantial excess of accumulation of the radiolabeledcompound in tumor as compared to both muscle and blood, and this ratioincreases over time.

In a second study, 14 mice were injected with [¹³¹I]-Compound 9 withincreasing amounts of unlabeled Compound 9. The mice were sacrificed at4 h p.i. and tissue analyzed as described above. While the addition of 2or 18 nanomoles of unlabeled Compound 9 decreased the amount of capturedlabel to some extent, it did not alter the tissue distribution to anysignificant extent.

In a third study a single mouse was injected with ^([124)I]-Compound 8and imaged with a microPET at around 3 and 17 hours post injection. Thearea of the tumor was plainly visible in the images obtained, along withresidual activity in the large intestine.

1. A compound of the formula:left side-linker−right side wherein (a) the left side has the formula:

in which: X₆ is NH₂; X₃ is hydrogen or halogen and X₁ is hydrogen, a C₁to C₁₀ alkyl, alkenyl, or alkynyl group, a C₁ to C₁₀ saturated orunsaturated hydrocarbon including a heteroatom in place of a carbonatom, or a targeting or labeling moiety, (b) the linker is selected fromthe group consisting of CH₂, O, NH and S, and (c) the right side has theformula:-six-membered aryl ring-(X₂)(X₄)(X₅) wherein X₂ is disposed at thetwo-position of the aryl ring and is halogen, alkyl, alkoxy, halogenatedalkoxy, hydroxyalkyl, pyrollyl, optionally substituted aryloxy,alkylamino, dialkylamino, carbamyl, amido, alkylamido dialkylamido,acylamino, alkylsulfonylamido, trihalomethoxy, trihalocarbon, thioalkyl,SO₂ -alkyl, COO-alkyl, NH₂, OH, CN, SO₂X₇, NO₂, NO, C═S R_(2,)NSO₂X_(7,), C═OR_(2,) where X₇ is F, NH₂, alkyl or H, and R₂ is alkyl,NH₂, NH-alkyl or O-alkyl; and X₄ and. X₅, which may be the same ordifferent, are disposed at the 4 and 5 positions of the aryl ring andwherein X₄ and X₅ are selected from halogen, alkyl, alkoxy, halogenatedalkoxy, hydroxyalkyl, pyrollyl, optionally substituted aryloxy,alkylamino, dialkylamino, carbamyl, amido, alkylamido dialkylamido,acylamino, alkylsulfonylamido, trihalomethoxy, trihalocarbon, thioalkyl,SO₂.alkyl, COO-alkyl, NH₂ OH, CN, SO₂X₅, NO₂, NO, C═SR₂NSO₂X_(5,),C═OR₂, where X₅ is F, NH2, alkyl or H, and R₂ is alkyl, NH₂, NH-alkyl orO-alkyl, C₁ to C₆ alkyl or alkoxy; or wherein X₄ and X₅ taken togetherhave the formula —O—(CH₂)_(n)—O—, wherein n is an integer from 0 to 2,or a pharmaceutically acceptable salt thereof.
 2. The compound of claim1, wherein X₄ and X₅ taken together have the formula —O—(CH₂)_(n)—O—,wherein n is an integer from 0 to
 2. 3. The compound of claim 2, whereinX₂ is NH₂, alkylamino or dialkylamino.
 4. The compound of claim 3,wherein X₁ is an amino alkyl moiety, optionally substituted on the aminonitrogen with one or two carbon-containing substituents selectedindependently from the group consisting of alkyl, alkenyl and alkynylsubstituents, wherein the total number of carbons in the amino alkylmoiety is from 1 to
 10. 5. The compound of claim 4, wherein X₁ includesa cyclic portion.
 6. The compound of claim 5, wherein the cyclic portioninclude at least one nitrogen.
 7. The compound of claim 4, wherein thelinker is S.
 8. The compound of claim 7, wherein X₁ includes a cyclicportion.
 9. The compound of claim 8, wherein the cyclic portion includeat least one nitrogen.
 10. The compound of claim 1, wherein X₁ is asaturated or unsaturated moiety containing up to 10 carbon atoms and atleast one heteroatom with substituents to satisfy valence.
 11. Thecompound of claim 10, wherein X₁ contains at least one nitrogenheteroatom.
 12. The compound of claim 10, wherein X₁ contains at leastone oxygen heteroatom.
 13. The compound of claim 10, wherein X₁ includesa cyclic portion.
 14. The compound of claim 11, wherein the cyclicportion includes at least one heteroatom.
 15. The compound of claim 11,wherein the cyclic portion is a 6-membered ring.
 16. The compound ofclaim 11, wherein the cyclic portion is a 3-membered ring.
 17. Thecompound of claim 10, X₄ and X₅ taken together have the formula—O—(CH₂)_(n)—O—, wherein n is an integer from 0 to
 2. 18. The compoundof claim 17, wherein X₂ is NH₂, alkylamino or dialkylamino.
 19. Thecompound of claim 1, wherein X₁ contains at least one nitrogenheteroatom.
 20. The compound of claim
 1. wherein wherein X₁ contains atleast one oxygen heteroatom.
 21. The compound of claim 1, wherein X₁includes a cyclic portion.
 22. The compound of claim 21, wherein thecyclic portion includes at least one heteroatom.
 23. The compound ofclaim 21, wherein the cyclic portion is a 6-membered ring.
 24. Thecompound of claim 21, wherein the cyclic portion is a 3-membered ring.25. The compound of claim 1, wherein the linker is CH₂.
 26. The compoundof claim 25, wherein X₄ and X₅ taken together have the formula—O—(CH₂)_(n)—O—, wherein n is an integer from 0 to
 2. 27. The compoundof claim 26, wherein X₂ is NH₂, alkylamino or dialkylamino.
 28. Thecompound of claim 27, wherein X₁ is an amino alkyl moiety, optionallysubstituted on the amino nitrogen with one or two carbon-containingsubstituents selected independently from the group consisting of alkyl,alkenyl and alkynyl substituents, wherein the total number of carbons inthe amino alkyl moiety is from 1 to
 10. 29. The compound of claim 28,wherein X₁ includes a cyclic portion.
 30. The compound of claim 29,wherein the cyclic portion include at least one nitrogen.
 31. Thecompound of claim 1, wherein the linker is S.
 32. The compound of claim31, wherein X₄ and X₅ taken together have the formula —O—(CH₂)_(n)—O—,wherein n is an integer from 0 to
 2. 33. The compound of claim 32,wherein X₁ includes a cyclic portion.
 34. The compound of claim 33,wherein the cyclic portion include at least one nitrogen.
 35. A methodof treating cancer and other aberrant biological condition where thecells depend on hsp90 activity for cell growth or maintenance in anindividual, comprising administering to the individual a therapeuticallyeffective amount of a compound in accordance with claim 1.